Tissue culture techniques. HeLa cells were infected with strains of parainfluenza

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1 STUDIES ON THE ANTIVIRAL ACTIVITY OF AMANTADINE HYDROCHLORIDE* Kenneth W. Cochran, Hunein F. Maassab, Akira Tsunoda, Byron S. Berlin Departmen[ OJ Epidemiology and Virus Laboratory, School of Public Health University of Michigan, Ann Arbor, Mich. Amantadine, or 1-adamantanamine hydrochloride, has been reported to possess activity against influenza virus in vitro, in mice and in man,*-4 and against rubella virus in v im5 The scope and mechanism of its activity, therefore, are of particular interest. This report describes the activity of amantadine against other respiratory viruses and offers new information concerning its site of action. Materials and Methods Tissue culture techniques. HeLa cells were infected with strains of parainfluenza viruses, types 2 (Greer) or 3 (C 243), obtained from R. M. Chanock, National Institutes of Health, Bethesda, Md. The LLC-MK2 line of monkey kidney cells6 was used for parainfluenza 3 and the WM strain of rubella viru~es.~ Other studies with rubella virus involved the chronically infected line previously described.8 The Long strain of respiratory syncytial virus, obtained from the American Type Culture Collection and grown in the H-L line of human epithelial cells, and the Japan 35 strain of influenza virus grown in primary calf kidney cells, were also used. Virus was detected by hemagglutination of guinea pig erythrocytes, by titration of infectivity, or by cytopathogcnic effect, depending on the particular cell-virus system. Antiviral activity was evaluated either by comparison of virus yield from treated and control cultures when measured by relative cytopathology, hemagglutination, or infectivity titrations, or by calculation of a therapeutic index? In vivo techniques. Swiss Webster mice or yearling ferrets were infected with influenza virus by a 3-minute exposure to aerosols of diluted suspensions of infected mouse lungs. The WR strain of vaccinia virus was given to mice intranasally. Disease was evaluated either by lung lesion scores1 or by mean survival times.' X-ray was given as previously described.11 Results Inhibition of myxoviruses in vitro. The problems of diagnosis and estimation of effectiveness make it important to assess the susceptibilities of all pneumotropic infectious agents to a drug proposed for any respiratory disease. In addition, the sharing of susceptibility to amantadine by several influenza viruses' and rubella virus,5 together with the noted similarities12 of rubella virus to the myxovirus group, prompted exploration of inhibition by amantadine as a property common to myxoviruses. Accordingly, the antiviral activity of amantadine for several additional myxoviruses was studied, with the results presented in TABLE 1. The HeLa, LLC-MK2 and HL cell lines maintained in our laboratory all tolerated 62.5 pg. of amantadine hydrochloride per ml., a concentration not directly virucidal to any of the viruses used in these experiments. Amantadine hydro- * These studies were aided by grants from Tbe National Foundation; the National Institute of Allergy and Infectious Diseases (A ); the Office of the Surgeon General, U. S. Army, under the auspices of the Commission on Influenza, Armed Forces Epidemiological Board; and by an Eli LiUy fellowship awarded to Dr. Tsunoda. 432

2 Cell-virus system Hemagglutinin, l/n Infectivity, 1-n Day afts virus Arnantadine HCl,,,g./ml. Amantadine HCl, rg./ml Parainfiuenza-2 vs HeLa, 1O:l (4+ 3+ I+) Parainfluenza-3 vs. HeLa, 1OO:l 16 Parainfluenza-3 vs. HeLa, 1O:l Parainfluenza-3 vs. LLC-MK2, 1O:l Resp. synct. vs. HL, 1O:l ( ) (CPE) (4+ 3+ I+) (CPE) (4+ 3+ I+) chloride in concentrations up to 62.5 pg.lml. did not inhibit hemagglutinin production in HeLa cells inoculated with parainfluema-2 virus at a multiplicity of 1: 1. However, when virus yield was measured by infectivity or cytopathology, there was inhibition, especially at the higher concentration. With parainfluenza 3 virus at a multiplicity of 1: 1, measurements of hemagglutinin or cytopathic activity did not disclose the inhibition seen in the production of infectious virus. At a lower multiplicity of parainfluenza 3 virus of 1: 1, all three measurementshemagglutination, infectivity, and cytopathology-revealed inhibition. With parainfluenza 3 virus in LLC-MK2 cells at a multiplicity of 1: 1, inhibition of virus production was observable whether measured by infectivity or hemagglutination. The ability of amantadine hydrochloride to inhibit respiratory syncytial virus was detectable by measurement of either the yield of infectivity or by cytopathology. These studies show that amantadine hydrochloride can inhibit selected myxoviruses other than influenza when used at high enough concentrations in a suitable host-virus system and with sensitive methods of following virus. Acrivity in mice and ferrets. Other experiments were directed toward exploring the activity of amantadine in viva Data showing the protective effect of amantadine hydrochloride against aerosol-induced influenza are presented in TABLE 2. It is noteworthy that the Lee strain of type B influenza was also susceptible. Vaccinia virus, included because of a recent report of its susceptibility in plaque inhibition tests,13 was given by intranasal injection. Vaccinia1 lung lesions were not found to be inhibited. The effectiveness of amantadine in vivo had been demonstrated only in mice' before its trial in man.2 Since more than one laboratory animal is usually desirable, amantadine was also tested for activity against influenza using ferrets. Amantadine hydrochloride is relatively more toxic for ferrets than for mice. Whereas 233 mg./kg. given intravenously is an LD5 for mice,l all ferrets receiv-

3 Average lung lesion score* Virus Treated? Control Iduenza, PR Iduenza, A,/AA/2/ Iduenza, B/Lee Vaccinia, WR Per cent decrease ing a single intraperitoneal dose of 2 mg./kg. of amantadine hydrochloride suffered convulsions and died in approximately 3 minutes. Daily intraperitoneal doses of 1 mg./kg. were tolerated for only two days, but given orally in two daily doses, the same amount was tolerated by ferrets for at least 13 days. Results of treating ferrets infected by aerosols of PR8 influenza virus are given in TABLE 3. From these results it may be seen that in ferrets amantadine hydrochloride aggravated rather than alleviated infection with influenza virus. Mechanism of action of amantadine: Influence of host immunity. Davies et a1.l showed the combination of amantadine with specific antiserum to be more effective against influenza in vitro than either treatment separately. This finding and other impres~ions~~ suggested that antibody might also participate in the antiviral activity of amantadine during the early stages of the in vivo infection. To test this hypothesis amantadine was studied in mice whose capacity for antibody production was impaired by a large sublethal dose of X-ray. Results of two experiments, given in TABLE 4, show that a single whole-body exposure to 35 R one day before virus, or treatment with amantadine hydrochloride starting two days before virus, provided protection as indicated by the increases in mean survival time. Results obtained with combined amantadine and X-ray treatment indicated TABLE 3 5,SQ x 13 5,SQ X 12 1, Oral x 6 3/3 Treated Control o/ o/ Fd6++++ /3 & Fd /3 Fd7 +++

4 Treatment Cochran et al.: Studies of Amantadine HCI 435 TABLE 4 SPARING EFFECT OF X-RAY AND AMANTADINE HCI ON PR 8 INFLUENZA MICE None ~ ~~~. X-ray? Amantadine HC1# Amantadine HC1+ x-ray Number of mice alive each day to day 17.' Mean survival* (days) 6.2, , , , 8.8 Total number of mice in group t 35 R, given in a single dose, one day before virus. t 1 or 5 mg./kg./day, subcutaneously X 2, starting two days before virus. that their individual effects were additive. Other data from appropriate control groups, not given in the Table, showed that amantadine had no significant influence on the radiosensitivity of mice. Locus of antiviral activity. On the basis of its time of effectiveness and comparison with specific antiserum, it was concluded' that the site of action of amantadine was the penetration by the virus of the host cell. The possibility that amantadine might be antagonizing an amino acid led to attempts to reverse its effect with several amino acids. Proline, tyrosine, phenylalanine, or tryptophane failed to alter the abil'ity of amantadine to inhibit influenza virus cytopathology in primary calf kidney cell cultures. Additional information was sought through comparison of the inhibitory activities of amantadine hydrochloride and aminophenylmethane sulfonic acid, the latter a type of inhibitor of influenza virus15 whose locus of action has been established to be early in the infectious cycle at adsorption or penetration.l6 The results, presented in TABLE 5, showed that in primary calf kidney cells infected with Japan 35 influenza virus, either amantadine hydrochloride or aminophenylmethane sulfonic acid was ineffective applied postinfection; that either was equally TABLE 5 CROSS TREATMENT OF INFLUENZA VIRUS (A,/JAPAN 35) WITH AMANTADIHE HYDROCHLORIDE AND AMINOPHENYLMETHANE SULFONIC ACID IN PRIMARY CALF KIDNEY CELL CULTURES Amantadee hydrochlonde (62.5 7/mI.) - Treatment. Aminophenylmethane sulfonic acid (25 v/ml.) Postinfection - - Postinfection Preinfection - - Preinfection Pre- and postinfection - - Pre- and postinfection Preinfection Postinfection Postinfection Preinfection

5 436 Annals New York Academy of Sciences effective used preinfection or combined pre- and postinfection. These data further show that inhibition due to either compound is maintained by the other. Although the inhibitory activity of substituted sulfonic acids has been associated with potassium in,17 added potassium ion did not reverse either aminophenylamethane sulfonic acid or amantadine hydrochloride in the calf kidney system. Development of resistance. Since amantadine hydrochloride had been found to inhibit acute rubella virus infection in ~ itro,~ was of interest to study its activity against the chronically infected cell line (RA) developed in this laboratory.8 The results, presented in TABLE 6, show that during four consecutive passages of RA cells chronically infected with rubella virus, treatment with amantadine hydrochloride did not reduce the third week virus yield. Subsequently, the persistence of rubella virus in amantadine-treated, chronically infected cultures was confirmed by immunofluorescence studies. This lack of inhibition in the chronic infection might have resulted from the virus becoming resistant during the prolonged growth period. However, when grown in several consecutive-treated acute infections, with treated virus used as the inoculum for the following passage, rubella virus retained its susceptibility to amantadine, exhibiting the same inhibition as previously untreated virus. Furthermore, when rubella virus from treated, chronically infected cells was used as the inoculum for the acute infection of LLC-MK2 cells, susceptibility was observed as indicated by a sixth-day virus titer of in untreated control cultures. Host cell Amantadine #K./ml. Int., 1- Passage I 1st 2nd 3rd 4th RA (chronic) LLC-MK, (acute) The observed maintenance of the susceptibility of rubella virus to inhibition by amantadine in vitro contrasted with the results seen with influenza. When influenza virus was grown in primary calf kidney cells in the presence of amantadine, the development of resistance to inhibition by amantadine was readily apparent, as shown in TABLE 7, where antiviral activity is presented as the therapeutic index.g These results show that influenza virus, initially susceptible to amantadine (therapeutic index = 16) or aminophenylmethane sulfonic acid (therapeutic index = 8) in one passage, albeit with several growth cycles, in the presence of either inhibitor lost its susceptibility to both inhibitors. Discussion The report by Davies et al. on the antiviral activity of amantadine hydrochloride' indicated a limited spectrum of activity among the myxoviruses. Their finding that influenza viruses of types A, A,, A*. C, and D (Sendai, para-

6 Cochran ef af.: Studies of Amantadine HCl 437 TABLE I DEVELOPMENT OF RESISTANCE TO AMANTADINE HCI AND AMINOPHENYLMBTHANE SULFONIC ACID BY INFLUENZA VIRUS (&/JAPAN 35) IN PRIMARY CALF KIDNEY CELL CULTURES Therapeutic index I vs. Amantadine vs. Aminophenylmethane HCl sulfonic acid Influenza, Japan 35 I l6 8 Influenza, Japan 35 passed 1 x 1 1 with amantadine HC1 Influenza, Japan 35, passed 1 x with aminophenylmethane sulfonic acid 1 1 influenza), together with the present results, indicate that all types of influenza virus can be susceptible. Our results with the parainfluenza and respiratory syncytial viruses suggest that the resistance of at least certain myxoviruses may be relative rather than absolute, and that susceptibility may not be evident depending on the methods and conditions used. The observed inhibition of infectivity without inhibition of hemagglutination may indicate incomplete virus. Some lines of given strains of influenza virus appear to differ in susceptibility, such as the lines of influenza B-Lee used by Davies et a/.' and by ourselves, as well as similar unpublished experiences of others. However, complicating any consideration of the inherent resistance or susceptibility of viruses to amantadine is the ability of at least one to develop resistance. It is significant that, although influenza virus readily developed resistance in vitro, we have yet to demonstrate the emergence of resistance to amantadine in vivo; specifically, in treated mice. In any event, amantadine does not engender sufficient resistance to prevent it from being effective in vivo, in contrast to guanidine and 2- (a-hydroxybenzy1)-benzimidazole, where resistance does vitiate their effectiveness in monkeys.18-2 Our present studies on the locus and mechanism of the antiviral activity of amantadine involve its use in comparison and conjunction with other agents of known activity. The application of X-ray to impair early immune responses indicated that specific antibody was not a necessary complement to amantadine activity in vivo. When amantadine was compared in vitro to aminophenylmethane sulfonic acid, an inhibitor of influenza virus with a known locus of activity, the two compounds were found to be essentially equivalent and interchangeable in their abilities to inhibit influenza virus and to induce mutual resistance. Neither inhibitor was reversed with selected amino acids.'5 Rubella virus, in contrast to influenza virus, retained its susceptibility to inhibition by amantadine, even when grown in chronically infected cells where its production was not decreased by amantadine treatment. Amantadine's similarity to aminophenylmethane sulfonic acid, which acts early in the cycle of virus replication during adsorption or penetration,i6 supports the earlier suggestion by Davies et a/.' that amantadine interferes with virus penetration, and is compatible with its decreased effectiveness in delayed treatment against rubella virus in vitro.5 Since the chronic in vitro rubella infection was not susceptible, the mode of action of amantadine enables one to conclude that the RA cell line8 represents a true chronic infection, maintained intracellularly as the cells divide, and not going through an amantadine-susceptible penetration phase. It is tempting to speculate

7 438 Annals New York Academy of Sciences that the development of resistance to amantadine by influenza virus and the persistence of susceptibility by rubella virus may be related to their immunologic mutabilities-influenza virus having the more variable surface, as reflected by its many and changing serotypes, compared with rubella virus's more stable surface and apparently single serotype. Amantadine attracted attention through its interesting structure, and seems to be maintaining its attraction in the current laboratory and early clinical testing phases. Its clinical testing requires time and experience, but in any event amantadine is making a significant contribution to the present, accelerating growth of interest and accomplishment, exemplified in part by this symposium, in the chemotherapy and chemoprophylaxis of viral diseases. Summary Amantadine hydrochloride at concentrations not toxic to host cells or free virus inhibited the growth of parainfluenza types 2 and 3 and respiratory syncytial viruses in vitro. In mice infected by virus aerosols, protection by amantadine was confirmed against A and A2 strains and was demonstrated against type B influenza. Vaccinial lung lesions in mice were not inhibited, while in ferrets amantadine treatment aggravated influenza. Amantadine protected both nonirradiated and irradiated mice and was additive with X-ray in exerting a sparing effect. The growth of rubella virus in chronically-infected LLC-MK2 cells was not inhibited by amantadine. This lack of inhibition was not due to the development of resistance by rubella virus, since acute infection of cells with virus produced in treated, chronically infected cells was susceptible. Rubella virus passed repeatedly in acutely infected, treated cells also retained susceptibility to amantadine. In contrast, influenza virus readily developed resistance in treated, acutely infected primary calf kidney cell cultures. When the inhibitory activity of amantadine hydrochloride was compared with aminophenylmethane sulfonic acid, a type of inhibitor acting early in the infectious cycle, both were found similarly effective in suppressing influenza virus production in vitro applied preinfection but not post-infection, and that inhibition due to either was maintained equally well by the other. Influenza virus made resistant to amantadine hydrochloride or aminophenylmethane sulfonic acid in vitro was also cross resistant to the other. This interchangeability for both inhibition and resistance was interpreted as indicating common sites of activity. References 1. DAVIES, W. L., R. R. GRUNERT, R. F. HAFF, J. W. MCGAWEN, E. M. NEUMAYER, M. PAULSHOCK, J. C. WAITS, T. R. WOOD, E. C. HERMANN & C. E. HOFFMANN Science 144: JACKSON, G. G., R. L. MULDOON & L. W. AKERS Antimicrobial Agents and Chemotherapy-1963 : WENDEL, H. A Federation Proc. 23: QUILLIGAN, JR., J. J. Antimicrobial Agents and Chemotherapy-1964 (in press). 5. MAASSAB, H. F. & K. W. COCHBAN Science 145: HULL, R. N., W. R. CHERRY &. J. TRITCH J. Exptl. Med. 115: VERONELLI, J. A., H. F. MAASSAB & A. V. HENNESSY Proc. SOC. Exptl. Biol. Med. 111: BROWN, G. C., H. F. MAASSALI, J. A. VERONELLI & T. FRANCIS, JR Science 145: KRAMER, P. E., M. L. ROBBINS & P. K. SMITH J. Pharmacol. Exptl. Therap. 113: 262.

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